Fluorescent proteins are useful in living cells and in vitro as components in reporters of gene expression, of cell abundance or location, of protein abundance or location, or of biochemical activities (Newman et al. (2011) Chem Rev 111:3614-3666). In these studies, researchers often desire to simultaneously assay two events, such as the expression of two genes, the abundance or location of two cell populations, the abundance and location of two proteins, or the occurrence and location of two biochemical activities (Depry et al. (2013) Pflugers Arch 465:373-381). This is possible with the use of two fluorescent protein labels of different colors. For instance, simultaneous dual-event imaging can be performed with a green fluorescent protein (GFP) and a red fluorescent protein (RFP) by exciting the GFP with blue or cyan light and exciting the RFP with green or yellow light. However, fluorescent proteins of different emission wavelengths usually have different excitation wavelengths, necessitating the use of two different excitation bands. This can be expensive when truly simultaneous excitation is needed, as it would necessitate a dual-bandpass filter in cases where wide-spectrum light sources are used, or two light-emitting diodes or lasers in cases where these single-wavelength light sources are used. In the case of two-photon-mode excitation, the Ti-Sapphire lasers used also are weaker in the wavelengths required for RFP excitation.
One strategy for reporting two biological processes with fluorescent proteins has been to use an RFP that can be excited by blue light (420-460 nm) together with cyan fluorescent proteins (CFPs), which are also excited by blue light (Kogure et al. (2008) Methods 45:223-226). Several blue-excitable RFPs, also termed long-Stokes-shift RFPs, have been developed and visualized simultaneously with CFP with blue excitation (Kogure et al. (2006) Nat Biotechnol 24:577-581; Piatkevich et al. (2010) PNAS USA 107:5369-5374; Yang et al. (2013) PLoS One 8:e64849). However, these RFPs have quantum yields of at most 0.27 (Table 1), far lower than the >0.6 of commonly used GFPs and CFPs (Lam et al. (2012) Nat Methods 9:1005-1012). Furthermore, the blue wavelengths required to excite them can cause phototoxicity and autofluorescence in cells due to their absorbance by flavin compounds. Furthermore, the vast majority of existing reporters use GFP, which is not well excited by blue wavelengths of light, rather than CFP, so imaging two biochemical activities using blue-excitable RFPs would usually require modification of existing GFP-based reporters to use CFP.
Another type of imaging in biological research that has been limited by technical performance has been bioluminescence imaging (BLI) in living animals. BLI refers to imaging of light produced by luciferase enzymes by oxidation of chemical substrates. In rodent preclinical studies, BLI performed on cells expressing non-secreted luciferases offers relatively cheap and simple means for in vivo tracking of cells, for example to assess stem cell survival or tumor growth (Close et al. (2011) Sensors (Basel) 11:180-206). For imaging in animals, luciferases that have high rates of production of red photons (above 600 nm) are preferred, as red light is capable of avoiding absorbance by hemoglobin and thereby transmits through tissue more readily. Much effort has thus been spent searching for luciferases with higher activity and redder emission. The second-generation firefly luciferase FLuc2 is currently most commonly used in animals, and has peak emission near 600 nm, but very low catalysis rates of 1.6 reactions per second with a bioluminescence quantum yield of 0.41 (Branchini et al. (1998) Biochemistry 37:15311-15319; Ando et al. (2007) Nature Photonics 2:44-47). Some non-secreted luciferases from marine organisms, such as those from Renilla and Cypridina species, have higher catalytic activities, but emit natively at wavelengths below 500 nm and with lower bioluminescent quantum yield (Shimomura et al. (1969) Science 164:1299-1300; Matthews et al. (1977) Biochemistry 16:85-91). Renilla luciferase has been engineered to emit at up to 550 nm (Loening et al. (2010) Nat Methods 7:5-6), but these wavelengths are still efficiently absorbed by hemoglobin. Nano luciferase (NLuc), a recently engineered version of a luciferase from the shrimp Oplophorus, has approximately 100× faster catalysis than FLuc2 (Shimomura et al. (1978) Biochemistry 17:994-998), but its output is even bluer, peaking at 460 nm, and it has not been demonstrated to improve detectability in vivo versus FLuc2 (Hall et al. (2012) ACS Chem Biol 7:1848-1857). In comparisons in animals, FLuc2 remained the most sensitive reporter for BLI imaging (Mezzanotte et al. (2013) Contrast Media Mol. Imaging 8:505-513).
Thus, there remains a need for fluorescent and bioluminescent proteins that improve detection and lower toxicity for use in fluorescence imaging and bioluminescence imaging.